This is an application for a K01 award for Dr. Vanessa Phelan, a chemist in the Department of Pharmacology at Skaggs School of Pharmacy and Pharmaceutical Sciences at the University of California, San Diego. Dr. Phelan is establishing herself as a young investigator in research of the metabolomics of polymicrobial biofilms. This K01 award will provide Dr. Phelan with the support necessary to accomplish the following goals: (1) to become expert at elucidating the chemical crosstalk in complex microbial communities; (2) to conduct clinically relevant investigations of the complex metabolic exchange within polymicrobial biofilms associated with cystic fibrosis lung infections; (3) to implement state-of-the-art mass spectrometry techniques to investigate mixed biofilms; (4) to develop a causal predictive model of the organisms and metabolites within biofilms and (5) to develop an independent research career. To achieve these goals, Dr. Phelan has assembled a mentoring team comprised of a primary mentor, Dr. Pieter Dorrestein, Associate Professor in Skaggs School of Pharmacy and Pharmaceutical Sciences, UCSD, who conducts academic research into metabolic exchange of microorganisms and one co-mentor: Dr. Suzanne Noble, an Assistant Professor in the Department of Medicine at University of California, San Francisco whose expertise in microbial genetics focuses her work on the interactions between C. albicans, co-isolates and the mammalian host. It is well established that microorganisms produce small molecules for both intra-species and inter- species interactions. However, the roles of these metabolites in the larger polymicrobial biofilm community remain uninvestigated. Dr. Phelan's research will focus on defining the metabolites responsible for establishing and maintaining a polymicrobial biofilm community derived of co-isolates from cystic fibrosis lung infections (Aim 1), the effect of perturbing these communities by removing key metabolites via gene knock-outs or introducing sub-inhibitory concentrations of antibiotics (Aim 2) and developing a predictive molecular network of the organisms (Aim 3). Dr. Phelan will use state-of-the-art mass spectrometry including two-dimensional and three-dimensional imaging mass spectrometry of microbial colonies and a new technique capable of sampling a microbial colony directly from a petri-dish. This research will form the basis for a multiplexed study of microbial biofilms includig proteomic, transcriptomic and metabolomic interactions to be proposed in an R01 grant application before the end of the K award.